Oriented biopolymeric membrane

ABSTRACT

A sheet membrane containing a layer of oriented biopolymeric fibers. The membrane has a thickness of 0.1 mm to 3.0 mm, a density of 0.1 g/cm 3  to 1.2 g/cm 3 , a hydrothermal shrinkage temperature of 50° C. to 85° C., a suture pullout strength of 0.1 kg to 5 kg, a tensile strength of 10 kg/cm 2  to 150 kg/cm 2 , and permeability to molecules having molecular weights of 200 to 300,000 daltons.

BACKGROUND OF THE INVENTION

[0001] Medical applications of biopolymeric membranes are manifold. See,e.g., Shu-Tung Li, Biologic Diomaterials: Tissue-Derived Biomaterials(Collagen). In: Biomedical Engineering Handbook, Ed. J. D. Bronzino,627-647, CRC Press, Inc. Boca Raton, Fla., 1995.

[0002] Biopolymeric membranes, such as collagen membranes, can be madeby air-drying a biopolymeric fibers-containing solution, or applying anacid or a base solution of dispersed biopolymeric fibers on a flatsurface. Li disclosed in U.S. Pat. No. 5,206,028 a method of preparing acollagen membrane by first freeze-drying a collagen dispersion to form asponge, which is then humidified, compressed, and subjected to chemicalcrosslinking. Chu et al., on the other hand, disclosed in U.S. Pat. No.4,725,671 a method of preparing a gel from an atelocollagen solution andthen compressing and air-drying the gel to form a collagen membrane.

[0003] The biopolymeric fibers in sheet membranes prepared by the priorart methods are randomly oriented. Such membranes generally have lowmechanical strength and are only useful in applications where mechanicalstrength of the device is not a critical factor for function. They arenot suturable and tend to tear with a slight suture tug. As most softtissue enforcement materials require extensive mechanical strength sothat they can be easily secured in place either by using sutures,staples, tags, or screws, mechanical strength becomes a critical factorin designing biopolymeric fiber-based membranes for applications in softtissue repair.

SUMMARY OF THE INVENTION

[0004] An aspect of this invention relates to a sheet membranecontaining at least one layer of oriented biopolymeric fibers, such ascollagen fibers. What is meant by “oriented” is that at least half ofthe biopolymeric fibers are in one general direction (i.e., “fiberorientation”) as determined by the method described below or by ananalogous method. The sheet membrane is generally flat but, if desired,can be somewhat curved. It has a thickness of 0.1 mm to 3.0 mm(preferably, 0.2 mm to 1.0 mm), a density of 0.1 g/cm² to 1.2 g/cm³(preferably, 0.2 g/cm³ to 0.8 g/cm³), a hydrothermal shrinkagetemperature of 50° C. to 85° C.(preferably, 55° C. to 70° C.), a suturepullout strength (both perpendicular and parallel to the fiberorientation) of 0.1 kg to 5 kg (preferably, 0.3 kg to 3 kg), and atensile strength of 10 kg/cm² to 150 kg/cm² (preferably, 30 kg/cm² to 80kg/cm²), and is permeable to molecules having molecular weights of 200to 300,000 daltons (preferably, 1,000 to 50,000 daltons). The aboverecited parameters can be readily measured by methods known to a personof ordinary skill in the art, some of which are described in detailbelow.

[0005] When a sheet membrane is made of two or more layers of orientedbiopolymeric fibers, the layers are secured to each other by fibringlue, collagen glue (gel or moist collagen sponge), suture (resorbableor nonresorbable), crosslinking formation, or the like. Preferably, thebiopolymeric fibers in different layers are respectively oriented indifferent directions.

[0006] Another aspect of this invention relates to a method of making asingle-layer sheet membrane of oriented biopolymeric fibers. The methodincludes: (1) reconstituting biopolymeric fibers, e.g., collagen fibers,dispersed in a solution; (2) placing the reconstituted biopolymericfibers around a mandrel; (3) rotating the mandrel to convert thereconstituted biopolymeric fibers on the mandrel into a tubular membraneof oriented biopolymeric fibers; (4) cutting the tubular membranelongitudinally after it has been dried on the mandrel; (5) rolling thecut membrane into a tubular form that is an inversion of the tubularmembrane; (6) inserting the rolled membrane into a tubular mesh; and (7)crosslinking the biopolymeric fibers to form a sheet membrane oforiented biopolymeric fibers.

[0007] Various medical uses of the sheet membranes of this invention aredescribed below. Other features or advantages of the present inventionwill be apparent from the following drawing and detailed description ofthe invention, as well as from the appending claims.

BRIEF DESCRIPTION OF THE DRAWING

[0008]FIG. 1 is a fabrication apparatus for orienting reconstitutedbiopolymeric fibers.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The membranes of the present invention contain at least one layerof biopolymeric fibers oriented in one direction, and therefore possessgreater mechanical strength and tear resistance. The improved propertiesallow such membranes to be sutured, stapled, tagged, or screwed intoplace to repair soft tissue.

[0010] Such membranes can be produced by dispersing biopolymeric fibersin an aqueous solution; reconstituting the dispersed fibers in onelayer; and orienting the reconstituted fibers. The membranes may includeselected bioactive agents such as growth factors, drugs, and the like.

[0011] Below are examples of how different membranes of this inventioncan be prepared.

[0012] A method of fabricating a reconstituted single-layer membrane ofthe present invention includes the following steps:

[0013] a) forming an aqueous dispersion containing biopolymeric fibers;

[0014] b) reconstituting the fibers;

[0015] c) orienting the reconstituted fibers on a rotating mandrel toform a tubular membrane;

[0016] d) compressing the hydrated fibers to remove excess solution;

[0017] e) drying the oriented fibers on the mandrel;

[0018] f) cutting the membrane perpendicular to the orientation of thefibers;

[0019] g) inverting the membrane; and

[0020] h) crosslinking the membrane

[0021] A method of fabricating a reconstituted two-layer membrane of thepresent invention includes the following steps:

[0022] a) dispersing fibers in an aqueous solution;

[0023] b) reconstituting the dispersed fibers;

[0024] c) orienting the reconstituted fibers on a rotating mandrel toform a tubular membrane;

[0025] d) compressing the hydrated fibers to remove excess solution;

[0026] e) drying the compressed fibers;

[0027] f) cutting the membrane perpendicular to the orientation of thefibers to form a sheet membrane;

[0028] g) placing around the sheet membrane a second sheet membraneprepared in the same manner;

[0029] h) inverting the two-layer membrane; and

[0030] i) crosslinking the membrane.

[0031] A method of fabricating a reconstituted three-layer membrane ofthe present invention includes the following steps:

[0032] a) dispersing fibers in an aqueous solution;

[0033] b) reconstituting the dispersed fibers;

[0034] c) orienting the reconstituted fibers on a rotating mandrel toform a tubular membrane;

[0035] d) compressing the hydrated fibers to remove excess solution;

[0036] e) overlaying a prefabricated membrane around the tubularmembrane on the mandrel;

[0037] f) orienting the reconstituted fibers again around theprefabricated membrane on the rotating mandrel;

[0038] g) compressing the hydrated fibers to remove excess solution;

[0039] h) drying the compressed fibers on the mandrel;

[0040] i) cutting the dried three-layer tubular membrane perpendicularto the orientation of the fibers to form a three-layer sheet membrane;

[0041] j) inverting the membrane; and

[0042] k) crosslinking the membrane.

[0043] Type I collagen fibers are preferred for preparing the membranesof the present invention due to its biocompatibility and easyaccessibility. Other biopolymeric materials, which can be either naturalor synthetic, include but are not limited to, other types of collagen(e.g., type II to type XXI), elastin, fibrin, polysaccharide (e.g.,chitosan, alginic acid, cellulose, and glycosaminoglycan), a syntheticanalog of a biopolymer by genetic engineering techniques, or acombination thereof.

[0044] Depending on the particular clinical application, orientation ofthe fibers in a membrane can be of particular importance. For example,in many surgical applications, a patch material is needed to enforce adiseased tissue. Thus, in repair of a hernia of the abdominal wall, anoriented membrane will provide a much higher strength than aconventional matrix. Also, a membrane used in periodontal and toothimplant surgeries has to be secured by sutures or tags, and an orientedmembrane, due to higher mechanical strength, will be more resistant totear than the conventional membrane. The membrane will also find itselfuseful as a sling patch in treating urinary incontinence conditions infemale patients. More specifically, an oriented membrane can serve as amechanical support to the bladder wall to minimize the mobility of theurethra and bladder neck. Other applications include use as a patch forpericardial tissue of heart and use as an aura repair patch.

[0045] Controlling the fiber orientation in a reconstituted membraneoptimizes the desired function of the membrane in vivo. Generally, thesuture pullout strength is higher in the direction perpendicular to thefiber orientation than in the direction parallel to the fiberorientation, whereas the tensile strength is stronger in the orientedfiber direction than the direction perpendicular to the fiberorientation. A membrane made of two or more layers of orientedbiopolymeric fibers affords an enhanced suture pullout strength andmechanical strength in the respective directions.

[0046] In particular, a collagen-based membrane of the present inventionmay be prepared by the following methods.

[0047] First, a native source of type I collagen, such as skin, bone,tendon, or ligament is cleaned, washed, and non collagenous impuritiesremoved by methods well known in the art such as that disclosed in U.S.Pat. No. 5,512,291 and in Oneson, et al., J. Am. Leather Chemists Assoc.65:440-450, 1970.

[0048] Next, a collagen dispersion is prepared. Generally, the purifiedcollagen material is dispersed in an acid solution. Either an organicacid such as acidic (CH₃COOH) or lactic acid CH₃CHOHCOOH) or aninorganic acid such as hydrochloric acid (Hcl) or sulfuric acid (H₂SO₂)may be used. The preparation of a collagen fiber dispersion is wellknown in the art such as those disclosed in U.S. Pat. Nos. 3,157,524 and5,326,350. These patents are included as references as if set out infull. The solid content of collagen fibers in the dispersion suitablefor the present invention is generally between 0.5% to 1.5%.Alternatively, a collagen dispersion may be prepared in an alkalinesolution.4 Sodium hydroxide (NaOH), potassium hydroxide (KOH), calciumhydroxide (Ca (OH)₂) are the common bases that may be used to preparethe alkaline dispersed collagen. If it is desirable to include abioactive molecule into the membrane, the bioactive molecule may bedissolved and mixed with the dispersed collagen in the dispersion.

[0049] An aliquot of acid dispersed collagen fibers is weighed into aflask. The dispersed collagen is reconstituted by neutralizing the acidwith a base (such as NH₄OH or NaOH) to a pH of about 5, the isoelectricpoint of this purified material. Other reconstituting agents such asneutral salts, non aqueous solvents or the like may be used toreconstitute the collagen fibers. The reconstituted, but still highlyhydrated, collagen fibers are oriented by winding the fibers onto arotating mandrel. Shown in FIG. 1 is an apparatus that is used forwinding the reconstituted collagen fibers. The apparatus 100 comprises amotor 101, an-adapter 102, a mandrel 103 fit into an adapter 102, and adispersion housing chamber 104. The reconstituted collagen fibers arefirst slowly poured into the chamber 104. The motor 101 with apredetermined speed is then turned on, causing the reconstituted fibers105 to be wound onto the mandrel 103 to form a tubular membrane.

[0050] The excess solution associated with the tubular membrane can beremoved by compressing the rotating mandrel against a smooth surfacesuch as a glass or a plastic sheet. The partially dehydrated, orientedmembrane is then dried. Depending on the desired permeability propertiesof the membrane, the drying can either be by air- or freeze-drying.Air-drying produces a membrane which allows the permeation of ions orsmall peptides (with molecular weight less than 2,000), whereas thefreeze-dried membranes permit the permeation of molecules ranging frommolecular weight from 200 to 300,000 (such as various growth factors andbioactive macromolecules). Desired permeability properties of themembranes can be obtained by controlling the extent of dehydration priorto freeze-drying.

[0051] The dried tubular membrane is then removed from the mandrel andcut along the long axis of the tube. The cut membrane is then invertedto a tubular form so that the inner (outer) wall of the original tubebecomes the inner (outer) wall. If necessary, the curvature of theinverted tube can be adjusted by creating an overlap between the twocutting edges or by leaving a gap between them. The inverted tube,having a reversed curvature, is inserted into a tubular mesh andcrosslinked with a crosslinking agent such as an aldehyde compound.Crosslinking of the inverted membrane under a certain reversed curvatureforces the membrane into a flat sheet geometry after crosslinking.Preferably, the tubular mesh is adjustable diameter so that it canaccommodate inverted membranes of all curvatures. Depending on thethickness of the membrane, a larger or smaller diameter tubular mesh maybe used. The tubular mesh may be constructed from biocompatible metalsor plastics (e.g. stainless steel and polypropylene).

[0052] The speed of rotation of the mandrel affects the degree oforientation of the collagen fibers in a given direction. Generally, ahigh speed of rotation of the mandrel (e.g., >700 rpm) generates ahigher degree of fiber orientation than a low speed rotation (e.g., <50rpm). Depending on the overall mechanical property requirements, thedegree of orientation can be adjusted by the speed of rotation of themandrel.

[0053] The degree of fiber orientation also depends on the diameter ofthe mandrel. All else being the same, a mandrel with a smaller diameterproduces a higher degree of fiber orientation. Preferably, the mandrelhas a diameter of about 1.0 cm to about 3.0 cm. However, other sizes mayalso be used if desired.

[0054] Another factor that contributes to the fiber orientation is theamount of reconstituted fibers per unit volume. The amount of collagenfibers per unit volume defines the thickness of the membrane of a givendiameter of the mandrel. Preferably, the amount of collagen fibers (dryweight) per cm length of a 1.25 cm-diameter mandrel is in the range ofabout 0.2 grams to about 0.8 grams. The thickness of the dry membraneproduced is in the range of about 0.2 mm to about 0.8 mm.

[0055] The degree of orientation can be determined by measuring andcomparing the acute angles of intersection between the fibers and afixed axis, e.g., the long axis of the tubular membrane. In order tofacilitate the determination of the angles of intersection, a dye suchas methylene blue may be used to stain the fibers and the acute anglesof intersection of various fibers with respect to the fixed axis canthen be easily measured with a protractor.

[0056] The extent of crosslinking determines the in vivo stability ofthe membrane. Depending on the functional requirements in vivo, theextent of crosslinking may be controlled accordingly. The extent ofcrosslinking in solution phase may be controlled by concentration,temperature, pH, and time of crosslinking. The crosslinking in vapor maybe controlled by vapor pressure, temperature, and time of crosslinking.

[0057] For membranes used to guide tissue regeneration in periodontaland tooth implant surgeries, it is desirable that the membranes bestable in vivo for about 8 to 16 weeks. For other applications such ashernia repair, sling patch, pericardial patch, or dura repair, the invivo stability is generally in the range of 6 to 12 months.

[0058] In vivo stability depends on the nature of the crosslinks formedby various crosslinking agents. Generally, glutaraldehyde forms morestable crosslinks than formaldehyde and carbodiimide. Thus,glutaraldehyde has been used to crosslink tissue heart valves for invivo durability, and formaldehyde has often been used to crosslinkresorbable implants.

[0059] The extent of crosslinking may be determined by methods wellknown in the art such as by monitoring the hydrothermal shrinkagetemperature or by determining the number of intermolecular crosslinks.In general, a hydrothermal shrinkage temperature in the range of 50° C.to 65° C. results in vivo stability for 8-16 weeks, and the hydrothermalshrinkage temperature in the range of 60° C. to 70° C. results in invivo stability for 12 to 24 weeks. For in viva stability greater than 6months, the shrinkage temperature should be tailored in the range of 70°C. to 85° C.

[0060] If it is desirable to have a specifically designed surface activemembrane, then chemical modification methods may be used to covalentlylink a bioactive molecule on the surface of the membrane. The surfacefunctional groups of collagen such as the side-chain amino groups oflysines and hydroxylysines, the side-chain carboxyl groups of asparticand glutamic acids, and the side-chain hydroxyl groups of hydroxyprolineand serines and threonines can be coupled with reactive functionalgroups of the bioactive molecules to form covalent bonds using couplingagents well known in the art such as aldehyde compounds, carbodiimides,and the like. Additionally, a spacer molecule may be used to gap thesurface reactive groups in collagen and the reactive groups of thebioactive molecules to allow more flexibility of such molecules on thesurface of the membrane.

[0061] In a two-layer membrane, the fiber orientations can be designedso as to enforce the mechanical properties in two directions.Specifically, a two-layer membrane is formed by overlaying aprefabricated layer on the top of another. By controlling the angle offiber orientations between the two layers, mechanical properties of thebilayer membrane are defined. The two layers can be secured to eachother by a biological glue such as collagen glue, fibrin glue, or thelike, or by sutures. The two layers can be further secured to each otherby crosslinking formation using crosslinking agents such as aldehydecompounds. The process can be repeated to produce as many layers asneeded, such that the final fiber orientation geometry and themechanical properties are strictly correlated and controlled.

[0062] Alternatively, a multi-layer membrane can be constructed directlyon the rotating mandrel. Using reconstituted fibers, a single-layermembrane is first cast on a rotating mandrel. A prefabricatedsingle-layer membrane sheet is then wrapped around the first membrane insuch a way so that the fiber orientations of the two membranes intersectat a desirable angle. A second membrane is then cast on the top of theoverlaid prefabricated membrane, forming a sandwich-like structure withcontrolled fiber orientations. If necessary, additional layers may beadded in an analogous manner. The process can be manipulated to producea variety of constructs with predetermined fiber orientations andmechanical properties. The multi-layer membranes can be secured bychemical crosslinking.

[0063] Without further elaboration, it is believed that one skilled inthe art can, based on the above description, utilize the presentinvention to its fullest extent. The following specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference.

[0064] Preparation of purified collagen fibers

[0065] The fat and fascia of bovine flexor tendon were carefully removedand washed with water. The cleaned tendon was frozen and commuted byslicing into 0.5 mm slices with a meat slicer. 1 kg of sliced wet tendonwas first extracted in 5 liters of distilled water at room temperaturefor 24 hours. The extractant was discarded and the 5 liters of 0.2 N HClin 0.5 M Na₂SO₄ was added and the tendon slices were extracted at roomtemperature for 24 hours. The acid solution was discarded and 5 litersof 0.5 M Na₂SO₄ was added to wash the tendon and to remove the residualacid. The acid extracted tendon was then extracted in 5 liters of 0.75 MNaOH in the presence of 1 M Na₂SO₄ at room temperature for 24 hours. Thebase solution was then discarded. The residual base was neutralized with0.01 N HCl to pH 5 followed by several changes of distilled water toremove the residual salts associated with the purified tendon. Thetendon was then defatted with isopropanol (tendon: isopropanol=1:5, v/v)for 8 hours at 25° C. under constant agitation. The extractant isdecanted and an equal volume of isopropanol was added and the tendonslices were extracted overnight at 25° C. under constant agitation. Thedefatted tendon was then dried under a clean hood. The purified collagenfibers were stored dry at room temperature for further processing.

[0066] Preparation of collagen fiber dispersions

[0067] A. Preparation of acid dispersed collagen fibers

[0068] Purified collagen fibers were weighed and dispersed in 0.07 Mlactic acid, homogenized with a Silverson Homogenizer (East Longmeadow,Mass.), and then filtered with a stainless steel mesh filter (40 mesh).The dispersion, which had a collagen content of 0.7% (w/v), wasdeaerated with vacuum to remove the trapped air.

[0069] B. Preparation of alkaline dispersed collagen fibers

[0070] Alternatively, purified collagen fibers were weighed anddispersed in 0.005 M NaOH, homogenized with a Silverson Homogenizer(East Longmeadow, Mass.), and then filtered with a stainless steel meshfilter (40 mesh). The dispersion, which had a collagen content of 1.0%(w/v), was deaerated with vacuum to remove the air trapped in it.

[0071] Fabrication of a single-layer oriented membranes

[0072] Acid dispersed collagen fibers (180 g) were reconstituted byadding 20 ml of 0.3% NH₄OH to its isoelectric point (pH 4.5-5.0). Thereconstituted fibers were poured into a fabrication apparatus with amandrel of 1.25 cm in diameter and were evenly distributed manually(FIG. 1). The fibers were oriented by rotating the mandrel at 250 rpm toform a tubular membrane. The excess solution was removed from thetubular membrane on the mandrel by compressing the membrane against twoglass plates. The partially dehydrated fibers on the mandrel werefreeze-dried (−10° C. for 24 hours, 20° C. for 16 hours at a pressureless than 200 millitorr) using a Virtis Freeze Dryer (Gardiner, N.Y.).The dried tubular membrane of fibers were cut along the longitudinaldirection, i.e., perpendicular to the fiber orientation. The cutmembrane was physically fixed in a sandwich form between twosemi-tubular stainless steel screens with the curvature of the membranereversed, and crosslinked with formaldehyde vapor generated from a 2%HCHO solution at 22° C. for 5 to 10 hours. The crosslinked membraneswere extensively rinsed in distilled water and freeze-dried.

[0073] Fabrication of a two-layer oriented membrane

[0074] A collagen glue was first prepared as follows: Alkaline dispersedcollagen fibers were freeze-dried under standard freeze dryingconditions (−10° C. for 48 hours, 20° C. for 16 hours at a pressure lessthan 200 millitorr) using a Virtis Freeze Dryer to form a sponge. Thefreeze-dried sponge was cut to the same size as the size of asingle-layer oriented membrane which had not been subjected tocrosslinking. The sponge was humidified for 8 hours at 25° C. with watervapor in a closed container. The humidified sponge was sandwichedbetween two uncrosslinked single-layer oriented membranes in such a waythat the orientation of one membrane was about 90° respect to that ofthe other membrane. The whole composite was compressed using amechanical press to form a cohesive membrane composite. The membrane wasthen crosslinked with HCHO vapor similar to that described above.

[0075] Alternatively, one crosslinked oriented membrane was overlaidover another with the fiber orientations of the two membranesintersecting at an angle of about 90 degrees. The two overlaid membraneswere sutured together using a 3-0 Dexon suture (Davis and Geck, Danbury,Conn.).

[0076] Fabrication of three-layer oriented membrane

[0077] Two humidified collagen sponges prepared in a manner describedabove were sandwiched between three uncrosslinked oriented collagenmembranes with the fiber orientations of the two top membranesintersecting at an angle of about 60 degrees and those of the two bottommembranes also at the same angle. The composite membrane was thencrosslinked in a manner described above.

[0078] Alternatively, three crosslinked oriented membranes were suturedtogether with a 3-0 Dexon suture.

[0079] Mechanical characteristics of oriented Membranes

[0080] A. Fiber Orientation

[0081] The fiber orientation of an oriented membrane of this inventionis determined by first staining the fibers with a dye material (such asmethylene blue for collagen fibers). The acute angle of intersectionbetween a reference line (e.g., a line corresponding to the long axis ofthe mandrel used to prepare the membrane) and a fiber can then bereadily measured. Such angles are measured for-a statisticallysignificant number of fibers. In each layer of an oriented membrane ofthis invention, the acute angles for at least 50±10% of the fibers, withrespect to the reference line, are within a relatively narrow range,i.e., ±30 degrees.

[0082] B. Thickness

[0083] The thickness of the membrane is determined with a caliper. Thethickness of a membrane of the present invention is generally within 0.1mm to 3.0 mm.

[0084] C. Density

[0085] To determine the density (g/cm³) of a membrane, the membrane isfirst dried under vacuum for 24 hours or over P₂O₅ for 24 hours and thedry weight is recorded. The dimensions (length, width and thickness) ofthe membrane are then measured with a caliper. Thus, the density is ameasure of the amount of per unit volume of the membrane. The density ofa membrane of the present invention is in the range of 0.1 g/cm³ to 1.2g/cm³.

[0086] D. Hydrothermal Shrinkage Temperature

[0087] A membrane having the dimensions 1.5 cm×2.0 cm is attached to ashrinkage temperature apparatus. See Li et al., Mat. Res. Soc. Symp.Proc. 331:25-32, 1994. The sample is first equilibrated in a beaker ofphosphate buffer saline (PBS). The solution is heated at a rate of 1° C.per minute. The length of the samples is continuously recorded. Thehydrothermal shrinkage temperature of the membrane is defined as thetemperature at which the length starts to change (onset point). Theshrinkage temperature of a membrane of this invention is in the rangefrom 50° C. to 85° C.

[0088] E. Mechanical Strength

[0089] Suture pullout strength perpendicular to fiber orientation:

[0090] The suture pullout strength of the wet membrane with suturepulling direction perpendicular to the fibers is determined with amechanical tester (Chatillon, Greensboro, N.C.). The membrane is cutalong the direction perpendicular to the fiber orientation to a size of20 mm×15 mm and soaked in phosphate buffered saline, pH 7.4 at 25° C.,for about 2 minutes. A suture (3-0 silk black braided, taper SH-1,Ethicon, Somerville, N.J.) is placed through the 20 mm membrane side atapproximately 4 mm from the edge. The suture is tied into a knot and issecured to the hook adapter of the tensile tester. The sample is thenclamped. The sample is pulled at a speed 1.0 in/min until the suture ispulled out. The suture pull out strength of a membrane of this inventionis in the range from 0.1 kg to 5.0 kg.

[0091] Suture pullout strength parallel to fiber orientation:

[0092] The suture pullout strength of the membrane having fibersparallel to the suture pulling direction is determined with a mechanicaltester (Chatillon, Greensboro, N.C.). The membrane is cut along thedirection parallel to the fiber orientation to a size of 20 mm×15 mm andsoaked in phosphate buffered saline, pH 7.4 at 25° C., for about 2minutes the test is performed as described above. The suture pull outstrength of a membrane of this invention is in the range from 0.1 kg to5.0 kg.

[0093] Tensile strength perpendicular to the fiber axis:

[0094] The mechanical strength of the wet membrane being pulled in thedirection perpendicular to the fibers is determined with a mechanicaltester (Chatillon, Greensboro, N.C.). The membrane is cut along thedirection perpendicular to the fiber orientation into a dumbbell shapewith a die punch. The sample is soaked in phosphate buffered saline, pH7.4, at 25° C. for about 2 minutes. The sample is then secured to aclamp fixture, and pulled at a speed 1.0 in/min until the sample ispulled apart. The tensile strength of a membrane of this invention is inthe range from 10 kg/cm² to 150 kg/cm².

[0095] Tensile strength parallel to the fibre axis:

[0096] The mechanical strength of the wet membrane being pulled in thedirection parallel to the fibers is determined with a mechanical tester(Chatillon, Greensboro, N.C.). The membrane is cut along the directionparallel to the fibre orientation into a dumbbell shape with a diepunch. The sample is soaked in phosphate buffered saline, pH 7.4 at 25°C., for about 2 minutes. The test is performed as described above. Thetensile strength of a membrane of this invention is in the range from 10kg/cm² to 150 kg/cm².

[0097] F. Permeability

[0098] A 2-cm diameter disk cut from a membrane of this invention isinserted into a hole between two compartments of a specially designedchamber, thereby completely separating the two compartments. A fixedvolume of PBS containing 50 μg of various sizes of peptide and proteinmolecules per ml is added to one compartment. The other compartment isfilled with a fixed volume of PBS only. The solutions in bothcompartments are allowed to equilibrate for 24 hours. An assay is thenconducted to determine the sizes of the peptide and protein molecules inthe compartment which initially only contains PBS. The membrane of thisinvention is permeable to molecules having molecular weights rangingfrom 200 to 300,000 daltons.

[0099] Use of oriented membranes in dental surgery

[0100] A. Periodontal surgery

[0101] Patients with advanced periodontitis are included in the study.More specifically, all of the patients have at least one pair of similarcontralateral periodontal lesions with probing depths of ≧5 mm andradiographic evidence of ≧40% bone loss. Each patient undergoes standardflap procedures, debridements, and bone depth measurements. Collagenmembranes of the present invention are then sized and adapted to thedefects and the flaps are replaced and sutured.

[0102] B. Tooth implant surgery

[0103] Patients suffer from tooth implant loosening are included. Foreach patient, the prosthesis is first removed and a full thickness flapreflected to allow adequate access to the affected area. The granulationtissue and other scar tissue are then carefully removed. The bone fillmaterial (autograft, allograft, or synthetic ceramics) is then placedinto the defect site and contoured to approximate ideal anatomy. Areconstituted collagen membrane of the present invention is trimmed tothe desired shape and placed snugly into the treated site. The membraneand bone fill material are secured using a bone tac kit (ImtecCorporation, Ardmore, Okla.) and the flap is rejoined with a standardsuture procedure.

[0104] Use of oriented membranes in repair of abdominal wall hernia

[0105] Adult mongrel dogs weighing 18 to 30 kg are used in this study.Under sterile conditions, a subtotal resection of the musculofacialportion of the abdominal wall of each dog is performed. The defect isrepaired with appropriately sized reconstituted collagen membrane of thepresent invention. The membrane is sutured by passing through fullthickness of the abdominal wall. The wound is then closed.

[0106] Use of Oriented Membrane in Female Incontinence Surgery

[0107] Female patients with genuine stress urinary continence arecandidates for the procedure. A midline anterior vaginal wall incisionis made from a level just below the external urethral meatus to a leveljust beyond the position of the Foley balloon. The retropubic space isentered on each side and blunt or sharp dissection, or both, is used togently free the paraurethral fascia (endopelvic fascia) from the lateralattachments to the inferior ramus of the pubic bone. A reconstitutedcollagen patch of the present invention is brought into the treated siteand applied to the suburethra and bladder base with No. 4 Dexon (Davisand Geck, Danbury, Conn.) sutures. A No. 0 suture is passed through thefour corners of the collagen patch. With each pass, the needle is alsotaken through the detached endopelvic fascia.

[0108] A 3-cm transverse suprapubic skin incision is then made and thepreviously placed sutures are passed through to the suprapubic areausing a Pereyra suspension needle. The vaginal incision is then closedwith running absorbable sutures and the sutures are tied above theanterior rectus fascia.

[0109] Use of Oriented Membrane in Dura Repair

[0110] Adult mongrel dogs weighing 18 to 30 kg are used in this study. Aleft frontoparietal craniotomy is carried out on each animal. A segmentof aura, measuring 4 cm×3 cm and overlaying the frontal and parietalcortex, is resected. Each alternate animal has a left frontal lobectomycarried out so that the graft will span an area of injured, as well asintact, cortex. A collagen membrane of this invention is sutured inplace by closely interrupted sutures of 5-0 mercilene. The bone flap isreinserted and muscle, fascia, and skin are closed with 2-0 sutures.

[0111] Other Embodiments

[0112] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A sheet membrane comprising a layer of orientedbiopolymeric fibers, wherein the membrane has a thickness of 0.1 mm to3.0 mm, a density of 0.1 g/cm³ to 1.2 g/cm³, a hydrothermal shrinkagetemperature of 50° C. to 85° C., a suture pullout strength of 0.1 kg to5 kg, and a tensile strength of 10 kg/cm² to 150 kg/cm², and ispermeable to molecules having molecular weights of 200 to 300,000daltons.
 2. The sheet membrane of claim 1 further comprising a secondlayer of oriented biopolymeric fibers secured to the first layer oforiented biopolymeric fibers, wherein the biopolymeric fibers of thefirst and second layers are respectively oriented in differentdirections.
 3. The sheet membrane of claim 2 further comprising a thirdlayer of oriented biopolymeric fibers secured to the second layer oforiented biopolymeric fibers, wherein the biopolymeric fibers of thefirst, second, and third layers are respectively oriented in differentdirections.
 4. The sheet membrane of claim 1, wherein the biopolymericfibers are collagen fibers.
 5. The sheet membrane of claim 4 furthercomprising a second layer of oriented collagen fibers secured to thefirst layer of oriented collagen fibers, wherein the collagen fibers ofthe first and second layers are respectively oriented in differentdirections.
 6. The sheet membrane of claim 5 further comprising a thirdlayer of oriented collagen fibers secured to the second layer oforiented collagen fibers, wherein the collagen fibers of the first,second, and third layers are respectively oriented in differentdirections.
 7. The sheet membrane of claim 1, wherein the membrane has athickness of 0.2 mm to 1.0 mm, a density of 0.2 g/cm³ to 0.8 g/cm³/ahydrothermal shrinkage temperature of 55° C. to 70° C., a suture pulloutstrength of 0.3 kg to 3 kg, and a tensile strength of 30 kg/cm² to 80kg/cm², and is permeable to molecules having molecular weights of 1,000to 50,000 daltons.
 8. The sheet membrane of claim 7 further comprising asecond layer of oriented biopolymeric fibers secured to the first layerof oriented biopolymeric fibers, wherein the biopolymeric fibers of thefirst and second layers are respectively oriented in differentdirections.
 9. The sheet membrane of claim 8 further comprising a thirdlayer of oriented biopolymeric fibers secured to the second layer oforiented biopolymeric fibers, wherein the biopolymeric fibers of thefirst, second, and third layers are respectively oriented in differentdirections.
 10. The sheet membrane of claim 7, wherein the membrane hasa thickness of 0.2 mm to 1.0 mm, a density of 0.2 g/cm³ to 0.8 g/cm³, ahydrothermal shrinkage temperature of 55° C. to 70° C., a suture pulloutstrength of 0.3 kg to 3 kg, and a tensile strength of 30 kg/cm² to 80kg/cm², and permeable to molecules having molecular weights of 1,000 to50,000 daltons.
 11. The sheet membrane of claim 10 further comprising asecond layer of oriented collagen fibers secured to the first layer oforiented collagen fibers, wherein the collagen fibers of the first andsecond layers are respectively oriented in different directions.
 12. Thesheet membrane of claim 11 further comprising a third layer of orientedcollagen fibers secured to the second layer of oriented collagen fibers,wherein the collagen fibers of the first, second, and third layers arerespectively oriented in different directions.
 13. The sheet membrane ofclaim 1 further comprising a bioactive agent.
 14. The sheet membrane ofclaim 4 further comprising a bioactive agent.
 15. The sheet membrane ofclaim 7 further comprising a bioactive agent.
 16. The sheet membrane ofclaim 10 further comprising a bioactive agent.
 17. A method of making asingle-layer sheet membrane of oriented biopolymeric fibers, said methodcomprising: reconstituting biopolymeric fibers dispersed in a solution;placing the reconstituted biopolymeric fibers around a mandrel; rotatingthe mandrel to convert the reconstituted biopolymeric fibers on themandrel into a tubular membrane of oriented biopolymeric fibers; cuttingthe tubular membrane longitudinally; rolling the cut membrane into atubular form that is an inversion of the tubular membrane; inserting therolled membrane into a tubular mesh; and crosslinking the biopolymericfibers, thereby forming a sheet membrane of oriented biopolymericfibers.
 18. The method of claim 17, wherein the biopolymeric fibers arecollagen fibers.
 19. The sheet membrane prepared by the method of claim17.
 20. The sheet membrane prepared by the method of claim 18.